Library construction method and reagent for pooling of large-sample-size PCR products based on high-throughput sequencing

Abstract

The invention discloses a library construction method and reagent for pooling of large-sample-size PCR products based on high-throughput sequencing. The library construction method comprises the following steps: separately subjecting each of a plurality of PCR products derived from DNAs of different samples to annealing with a random primer having a tag sequence and carrying out an isothermal amplification reaction under the action of an isothermal amplification enzyme; mixing isothermal amplification products derived from different samples and carrying out piece selection on the mixed products; subjecting the products of the piece selection to repairing of 5' phosphorylated blunt ends and A addition reaction of 3' ends; connecting the products with linker sequences so as to obtain ligation products capable of distinguishing library information; and subjecting the ligation products to PCR amplification to obtain a library applicable to high-throughput sequencing. The method provided bythe invention realizes pooling without dependence on an ultrasonic breaking instrument, is lowered in library construction cost and complexity, reduces data waste, and improves the base randomness and coverage depth uniformity of sequencing data, thereby reducing the sequencing cost of a single sample.

Description

Technical field [0001] The present invention relates to the technical field of library construction, in particular to a method and reagents for library construction based on high-throughput sequencing-based PCR product large sample volume mixed library construction. Background technique [0002] With the development of sequencing technology, gene sequencing has entered the age of thousands of genomes, but the sequencing and analysis costs of whole genomes are still very expensive. Therefore, the capture and sequencing of exon regions or specific regions of interest is more in line with actual needs. For the construction of next-generation sequencing libraries of a few or dozens of target gene amplification sequences, single-tube amplification and then mixed library construction or multiplex PCR are often used. Since the target area of ​​each sample is small and the data demand is low, it is generally necessary to perform mixed sequencing on hundreds or thousands of samples. In t...

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Problems solved by technology

The above method is simple to operate, but there are problems in the following aspects: one is that after the ultrasound is interrupted, some inserted fragments lose the label sequence that distinguishes the sample information, so some invalid data will be generated, resulting in a waste of data; the other is to realize the sequence Full coverage, paired-end sequencing must be used, the size range of insert fragments is large, and the quality of the library is relatively poor, which affects the quality of off-machine data; the third is to rely on ultrasonic interruption equipment

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